Spongiform encephalopathies are transmissible diseases that can have a major economic impact on agricultural exports, and pose a significant challenge for surveillance of the food supply. Scientists generally believe that these diseases are transmitted via a self-propagating, aberrant conformation of the prion protein (PrP). This prion hypothesis suggests that PrP alone should be sufficient to cause symptoms or death. If this hypothesis is true, then it should be possible to reproduce the disease using recombinant proteins expressed in yeast or bacteria. In tomorrow’s Science, researchers from Columbus and Shanghai report that they have managed to do this, establishing that PrP alone can account for prion disease transmission.

Previously, other groups had successfully produced recombinant PrP (recPrP) and generated amyloid fibrils that appeared to contain the pathogenic conformation (PrPSc). When injected into mice, however, these amyloids had limited infectivity, which raised doubts as to whether these fibers are the cause of the disease. Wang et al. decided to take a different route, using a technique known as protein misfolding cyclic amplification (PMCA). In this approach, misfolded aggregates of a protein are broken up using sound waves and then incubated with normal, folded protein. If the misfolded protein can cause normal protein to adopt an aberrant conformation (as PrPSc can), then the misfolded protein will be amplified. By performing many cycles of this experiment, one can in principle produce a very large amount of PrPSc from a single misfolded chain.

Wang et al. also added some ingredients to their reactions that they believed would promote prion formation: RNA and a lipid called POPG. Under these conditions, they detected a protease-resistant protein after 17 rounds of PMCA amplification. Under normal circumstances, PrP is cleaved by the protease, like a pair of scissors cutting a string. If PrP has aggregated, however, it becomes much more difficult to cut, as if you were using safety scissors to cut a thick hemp rope. The researchers discovered that the protein from this reaction (which they called rPrP-res) could cause normal protein to also become protease resistant. And, when they treated protein from mouse brains with rPrP-res, they found that it, too, formed protease-resistant aggregates.

To really put the hypothesis to the test, however, tests in live animals were needed. The researchers injected one group of animals with the product of a PMCA seeded with rPrP-res. They also injected three additional groups of animals with control cocktails to prove that neither the non-protein ingredients, nor the unprocessed protein, caused the disease. None of the control animals developed symptoms of encephalopathy during the experience, but all of the mice injected with the PMCA product died in about five months, while displaying clinical signs of prion disease. Their brains were inspected post-mortem using histological and molecular means. The brains clearly showed the formation of the vacuoles that give spongiform encephalopathy its name. Additionally, protease-resistant PrP was detected in homogenates of the brain tissue, indicating that the rPrP-res had propagated in the living mouse brains, causing disease and eventual death. When these homogenates were injected into the brains of other healthy mice, a similar pattern of pathology recurred. This proved that the effects of rPrP-res could be serially propagated, just as prion disease is.

These results leave little room for doubt that misfolded PrP is sufficient to cause prion disease; no other infectious agent was required. The effectiveness of RNA and POPG in promoting the pathogenic conformation may indicate that these or similar molecules play a role in the spontaneous development of prion disease. Aside from adding to our knowledge directly, this research has the potential to significantly increase our ability to investigate prion disease. The ability to produce bona fide infective prion molecules in vitro from recombinant protein opens up new avenues for experiments in structural biology and biochemistry that may enable us to cure or entirely prevent these diseases, rather than just trying to contain them.